RESUMO
The marine yeast Debaryomyces hansenii is of high importance in the food, chemical, and medical industries. D. hansenii is also a popular model for studying molecular mechanisms of halo- and osmotolerance. The absence of genome editing technologies hampers D. hansenii research and limits its biotechnological application. We developed novel and efficient single- and dual-guide CRISPR systems for markerless genome editing of D. hansenii. The single-guide system allows high-efficiency (up to 95%) mutation of genes or regulatory elements. The dual-guide system is applicable for efficient deletion of genomic loci. We used these tools to study transcriptional regulation of the 26S proteasome, an ATP-dependent protease complex whose proper function is vital for all cells and organisms. We developed a genetic approach to control the activity of the 26S proteasome by deregulation of its essential subunits. The mutant strains were sensitive to geno- and proteotoxic stresses as well as high salinity and osmolarity, suggesting a contribution of the proteasome to the extremophilic properties of D. hansenii. The developed CRISPR systems allow efficient D. hansenii genome engineering, providing a genetic way to control proteasome activity, and should advance applications of this yeast.
Assuntos
Sistemas CRISPR-Cas , Debaryomyces/enzimologia , Debaryomyces/genética , Edição de Genes/métodos , Complexo de Endopeptidases do Proteassoma/genética , Saccharomyces cerevisiae/genética , Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Extremófilos/enzimologia , Extremófilos/genética , Regulação da Expressão Gênica , Genoma Fúngico , Organismos Geneticamente Modificados , Osmorregulação/genética , Estresse Oxidativo/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Salino/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Halotolerant species are adapted to dealing continually with hyperosmotic environments, having evolved strategies that are uncommon in other organisms. The HOG pathway is the master system that regulates the cellular adaptation under these conditions; nevertheless, apart from the importance of Debaryomyces hansenii as an organism representative of the halotolerant class, its HOG1 pathway has been poorly studied, due to the difficulty of applying conventional recombinant DNA technology. Here we describe for the first time the phenotypic characterisation of a null HOG1 mutant of D. hansenii. Dhhog1Δ strain was found moderately resistant to 1 M NaCl and sensitive to higher concentrations. Under hyperosmotic shock, DhHog1 fully upregulated transcription of DhSTL1 and partially upregulated that of DhGPD1. High osmotic stress lead to long-term inner glycerol accumulation that was partially dependent on DhHog1. These observations indicated that the HOG pathway is required for survival under high external osmolarity but dispensable under low and mid-osmotic conditions. It was also found that DhHog1 can regulate response to alkali stress during hyperosmotic conditions and that it plays a role in oxidative and endoplasmic reticulum stress. Taken together, these results provide new insight into the contribution of this MAPK in halotolerance of this yeast.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana Transportadoras/genética , Osmorregulação/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Álcalis/efeitos adversos , Regulação Fúngica da Expressão Gênica , Glicerol/metabolismo , Pressão Osmótica/fisiologia , Fosforilação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Saccharomycetales/fisiologia , Transdução de Sinais/genéticaRESUMO
Debaryomyces hansenii is a halotolerant yeast of importance in basic and applied research. Previous reports hinted about possible links between saline and oxidative stress responses in this yeast. The aim of this work was to study that hypothesis at different molecular levels, investigating after oxidative and saline stress: (i) transcription of seven genes related to oxidative and/or saline responses, (ii) activity of two main anti-oxidative enzymes, (iii) existence of common metabolic intermediates, and (iv) generation of damages to biomolecules as lipids and proteins. Our results showed how expression of genes related to oxidative stress was induced by exposure to NaCl and KCl, and, vice versa, transcription of some genes related to osmotic/salt stress responses was regulated by H2O2. Moreover, and contrary to S. cerevisiae, in D. hansenii HOG1 and MSN2 genes were modulated by stress at their transcriptional level. At the enzymatic level, saline stress also induced antioxidative enzymatic defenses as catalase and glutathione reductase. Furthermore, we demonstrated that both stresses are connected by the generation of intracellular ROS, and that hydrogen peroxide can affect the accumulation of in-cell sodium. On the other hand, no significant alterations in lipid oxidation or total glutathione content were observed upon exposure to both stresses tested. The results described in this work could help to understand the responses to both stressors, and to improve the biotechnological potential of D. hansenni.